Integrins are glycoprotein cell adhesion receptors that mediate cell-cell (via counter receptors on other cells) and cell-substrate (i.e. cell-extra cellular matrix) interactions [Aplin at al., Pharmacological Reviews 50:199-252 (1998)]. Integrins function generally to maintain tissue integrity, cellular migration, provide physical support for cells, allow for cohesion between cells, permits the generation of traction forces which enable movement, and to organize signaling complexes which modulate differentiation, cell fate, and apoptosis. Additionally, the properties of the individual integrins depend upon which subunits it contains and which cells they are expressed in. For example, the leukocyte integrins mediate several adhesive events that are crucial for immune system function. They promote the adhesion that is required for T lymphocyte target cell lysis [Davignon, et al., Proc. Natl. Acad. Sci. USA 78:4535-4539 (1981)], T lymphocyte proliferation [Davignon, et al., Proc. Natl. Acad. Sci. USA 78:4535-4539 (1981)], natural killing [Krensky et al., J. Immunol 131:611-616 (1983)], leukocyte adhesion to, and migration through endothelial cells [Dustin et al., J. Cell Biol. 107:321-331 (1988); Harlan et al., Blood 66:167-178 (1985); Haskard et al, J. Immunol. 137:2901-2906 (1986); Lo et al., J. Exp. Med. 169:1779-1793 (1989); Lo et al., J. Immunol. 143(10):3325-3329 (1989); Smith, et al., J. Clin. Invest. 83:2008-2017 (1989); Smith, et al., J. Clin. Invest. 82:1746-1756 (1988)], neutrophil homotypic aggregation, and neutrophil chemotaxis [Anderson, et al., J. Immunol. 137:15-27 (1986)].
Each integrin is a heteroduplex consisting of an alpha subunit and a beta subunit. There are currently 19 different alpha subunits and 8 different beta subunits, perhaps making the integrins the most structurally and functionally diverse family of cell adhesion molecules [e.g. see Springer, T. A. Nature 346:425-433(1990); Smyth et al., Blood 81:2527-2843 (1993); Springer, T. A. Proc. Natl. Acad. Sci. 94:65-72 (1997); Humpries, M. J. Biochem. Soc. Trends 28(4):311-339 (2000)].
Each subunit contains a transmembrane domain anchoring the major portion of the wild-type protein to the external side of the cell's membrane.
Each subunit has a large extracellular domain, a single transmembrane domain and usually a relatively short transmembrane domain [Aplin at al., Pharmacological Reviews 50:199-252 (1998)]. The pairing of a particular alpha subunit with a particular beta subunit in part determines the ligand-binding characteristics of the integrin protein. As such, both of the subunits can alter the binding characteristics of the integrin protein. Studies of integrin binding characteristics have focused on three areas: a) a series of seven repeats near the N-terminal portion of the alpha subunit, b) an inserted domain (I-domain also known as an “A domain”) in the alpha subunit, and c) an “I-domain like region” located in the beta subunit [(Loftus & Liddington, J. Clin. Invest. 99:2302-2306 (1997)].
The functional integrin protein appears to exist in two different states, open (activated or high affinity) and closed (“inactivated” or “low affinity”). The open state allows the integrin protein to bind to its appropriate ligand, while the closed state is relatively inert. The ability of integrins to bind to ligands depends upon internal cell messages, as well as the presence or absence of divalent cations such as Mg+2 or Ca+2 [(Springer, Proc. Natl. Acad. Sci. 94:65-72 (1997)]. This regulation of binding affinity integrin is believed to be due to the different conformational states that integrin can exist in. Signals which after this conformation, either internal cell signals or divalent cations, bias the stability of each conformational state of integrin into an “open” or “closed” conformation.
The leukocyte integrin subfamily includes four members, LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150,95 (CD11c/CD 18), and alphaDBeta2 that share a common.beta subunit that is noncovalently associated with unique but closely related alpha chains [Kishimoto et al., Adv. Immunol. 46:149-182 (1989); Springer, Nature 346:425-433 (1990)]. These glycoproteins share a common CD18 beta subunit (95,000 MW) but have individual unique CD11 alpha subunits (175,000, 160,000, 150,000 MW) respectively, that are structurally homologous [Larson, et al., J. Cell Biol. 108:703-712 (1989)]. All four members share two prominent features in the extracellular region of the molecule, a putative divalent cation binding region consisting of three tandem repeats of an EF-hand motif, and approximately a 200 amino acid inserted or “I” domain [Arnaout, et al., J. Cell Biol. 106:2153-2158 (1988); Corbi, et al., J. Biol. Chem. 263:12403-12411 (1988); Corbi, et al., EMBO J. 6:4023-4028 (1987); Kaufman, et al., J. Immunol. 147:369-371 (1991); Larson, et al., J. Cell Biol. 108:703-712 (1989); Pytela, EMBO J. 7:1371-1378 (1988)].
Mac-1 plays a central role in promoting neutrophil inflammatory responses, and its use as a target in medical research has shown promise in treating autoimmune diseases and ischemia/reperfusion. It is expressed on the cell surface as well as in an intracellular, vesicular compartment in circulating neutrophils and monocytes which is mobilized to the cell surface by inflammatory mediators [Todd, et al., J. Clin. Invest. 74:1280-1290 (1984); Springer, et al., In: Biochemistry of Macrophages (CIBA Symposium 118), Pitman, London, pp. 102-126 (1986); Lanier, et al., Eur. J. Immunol. 15:713-718 (1985); Yancey, et al., J. Immunol. 135:465-470 (1985)].
The I domain of Mac-1 contains 184 residues and is implicated in the integrin's binding to protein ligands [Michishita et al., Cell 72:857-867 (1993); Diamond et al., J. Cell Biolo. 120:1031-1043 (1993); Lee et al., Structure 3:1333-1340]. Mac-1 can bind to iC3b, intercellular adhesion molecule-1 (ICAM-1, ICAM-2 and fibrinogen) as well as Factor X. I domains in the broader category of integrins may also bind to various collagen isotypes (I and IV) as well as laminin. [Humphries, Biochemistry Society 28:311-339 (2000)]. Crystal structures of I domains reveal a dinucleotide-binding fold, with a metal ion-dependent adhesion site (MIDAS) on the top face [Lee et al. Structure 3:1333-1340 (1995); Lee et al. Cell 80:631-638 (1995); Qu & Leahy, Proc. Natl. Acad. Sci. U.S.A. 92:10277-10281 (1995); Qu & Leahy, Structure 4:931-942 (1996); Emsley et al. J. Biol. Chem. 272:28512-28517 (1997); Baldwin et al., Structure 6:923-935 (1998); Nolte et al., FEBS Lett. 452:379-385 (1999); Rich et al., J. Biol. Chem. 274:24906-24913 (1999)]. The metal ion ligates an acidic residue in protein ligands, and is surrounded by residues that contact the ligand [Lee et al., Structure 3:1333-1340(1995); Huang and Springer, J. Biol. Chem. 270:19008-19016 (1995); Li et al., J. Cell Biol. 143:1523-1534(1998); Zhang et al., Biochemistry 38:8064-8071 (1999)]. The bottom of the I domain connects to a putative integrin beta-propeller domain [Springer, Proc. Natl. Acad. Sci U.S.A. 94:65-72 (1997)].
Two different crystal forms of the Mac-1 I domain, termed open and closed, respectively, are hypothesized to represent the I domain in active (or ligand binding) and inactive (ligand nonbinding) conformations. [Lee et al., Structure 3:1333-1340 (1995); Lee et al., Cell 80:631-638 (1995)]. Although experimental data support this idea [Li et al., J. Cell Biol. 143:1523-1534 (1998); Oxvig et al., Proc. Natl. Acad. Sci. U.S.A. 96:2215-2220 (1999)] it has remained controversial because many other I-domain structures, including those from other alpha subunits, have failed to reveal a corresponding open conformation. [Qu et al., Proc. Natl. Acad. Sci. U.S.A. 92:10277-10281 (1995); Qu et al., Structure 4:931-942 (1996); Emsley et al., J. Biol. Chem. 272:28512-28517 (1997); Baldwin et al., Structure 6:923-935 (1998); Nolte et al., FEBS Lett. 452:379-385 (1999) Rich et al., J. Biol. Chem. 274:24906-24913 (1999)]. However, a recent co-crystal of the alph2 I domain bound to a triple-helical collagen peptide ligand reveals an open conformation very similar to that described for alpha M. [Emsley et al., Cell 101:47-56 (2000)]. Between the closed and open structures, three residues that directly coordinate the metal differ, in position, and other nearby residues shift in position. These movements appear to be structurally linked to a dramatic, 10 Å movement in the C-terminal alpha helix. The structurally homologous G-protein alpha subunit undergoes a similar change in metal coordination between the GDP- and GTP-bound forms, which is coupled to long-range structural rearrangements [Lee et al., Structure 3:1333-1340 (1995)].
One of the ligand binding sites for Mac-1 is believed to be near MIDAS. [Huang & Springer J. Biol. Chem. 270:19008-19016 (1995)].
Mutations that stabilize one protein conformation relative to another have previously been found empirically, for example in hemoglobin [Perutz, Q. Rev. Biophys 22:139-237 (1989)]; furthermore, visual inspection by experts has been used to predict mutations that stabilize the open conformer of the Mac-1 I domain [Li et al., J. Cell Biol. 143:1523-1534 (1998)].
Recently, advances have been made in computational design. Several groups have applied and experimentally tested systematic, quantitative methods to protein design with the goal of developing general design algorithms (Hellinga et al., J. Mol. Biol. 222: 763-785 (1991); Hurley et al., J. Mol. Biol. 224:1143-1154 (1992); Desjarlaisl et al., Protein Science 4:2006-2018 (1995); Harbury et al., Proc. Natl. Acad. Sci. U.S.A. 92:8408-8412 (1995); Klemba et al., Nat. Struc. Biol. 2:368-373 (1995); Nautiyal et al., Biochemistry 34:11645-11651 (1995); Betzo et al., Biochemistry 35:6955-6962 (1996); Dahiyat et al., Protein Science 5:895-903 (1996); Dahiyat et al., Science 278:82-87 (1997); Dahiyat et al., J. Mol. Biol. 273:789-96; Dahiyat et al., Protein Sci. 6:1333-1337 (1997); Jones, Protein Science 3:567-574 (1994); Konoi, et al., Proteins: Structure, Function and Genetics 19:244-255 (1994)). These algorithms consider the spatial positioning and steric complementarity of side chains by explicitly modeling the atoms of sequences under consideration. In particular, WO98/47089, and U.S. Ser. No. 09/127,926 describe a system for protein design; both are expressly incorporated by reference. With the assistance of these programs mutations have been designed that enhance the stability of small proteins (on the order of 60 residues) [Dahiyat et al., Science 278:82-87 (1997); Malakauskas, & Mayo, Nature Struc. Biol. 5:470-475 (1998)].
Because of the huge functional difference between the two states of the integrin protein, substances which bias one state of the protein over another can provide an effective method of altering the concentration and activity of integrin and dealing with any integrin related problems. Additionally, the ability to monitor the various states that these proteins exist in a state dependent manner is also possible due to the current invention because known populations of single state integrins may be screened against possible probes selective only for that state.
Accordingly, it is an object of the invention to provide conformationally biased integrins for the treatment of diseases in which integrins have been implicated, including but not limited to: autoimmune diseases, inflammatory diseases, transplant rejections, apoptosis, and various forms of shock (i.e. hypovolemic or cerebral), the existence of such conformationally biased proteins will enable more effective drug and antibody design to help with these disorders.